The functions that microtubules serve in cells require explanations for the mechanisms that determine the initiation, growth, directionality, and spatial localization of these structures. The principal objectives of this proposal are three-fold. (1) Biochemical studies will be continued to elucidate the molecular mechanism of the in vitro self-assembly of microtubules in terms of the specific chemical steps that occur between the depolymerized state and the fully assembled structure. The nucleation of assembly, role of microtubule-associated proteins, participation of guanine nucleotides and possible head-to-tail mechanism of assembly will be investigated. (2) Possible mechanisms for both the temporal and spatial regulation of microtubule assembly will be explored. Temporal mechanisms to be studied include control mediated through calcium ions, through pools of active and inactive tubulin subunits, and through microtubule-associated proteins. Spatial mechanisms will involve determining the nature, activity, and regulation of the material at microtubule organizing centers responsible for the positional localization of microtubules. The microtubule organizing centers to be investigated are the centrosomes and kinetochores of Chinese hamster ovary cells, the centrospheres of sea urchin eggs, and the spindle-pole bodies of yeast. (3) Aspects of the formation and function of the mitotic spindle will be studied by (a) determining the growth polarity of microtubules initiated by centrosomes and chromosomes, and (b) attempting to develop an in vitro model for mitosis using oocytes of the marine worm Chaetopterus.